Transcriptional control allows coordinated expression of genes involved in similar processes or life stages. Entamoeba histolytica is a leading cause of parasitic death, but little is known about the virulence program that this protozoan parasite uses to cause invasive colonic or hepatic disease. Although a great deal of information is available on transcriptional regulatory mechanisms in model organisms, there is limited knowledge about such mechanisms in most parasite systems. The long-term goals of our lab are to understand how parasite genetics and gene expression affect virulence. Our goal is to identify transcriptional regulatory networks that control expression of virulence associated gene subsets in E. histolytica. The identification of transcriptional regulatory mechanisms in E. histolytica will give an insight into the basic biology of this parasite, as well as provide avenues for studying the regulation of amebic genes. In Aim 1 we will identify promoter motifs that allow coordinated expression of genes involved in virulence. Additionally, we will look for conserved motifs in the 3'UTR and downstream genomic regions of the gene sets of interest. Our definition of "virulence" includes the cysteine proteinase genes (involved in tissue invasion), genes involved in the stress response in ameba (Ehssp gene family), genes differentially expressed in virulent and non-virulent E. histolytica strains, genes regulated in vivo in an animal model of colitis, and genes regulated during parasite development. In Aim 2, we will functionally characterize potential regulatory motifs from the cysteine proteinase, Ehssp, and the strain-specific gene families using reporter gene and electrophoretic mobility shift assays. Success in these aims will allow us to show proof of concept that our approach is valid. Motifs that control expression of genes regulated during an animal model of colitis and during development will be characterized at a later date as those are technically much more challenging and will require the development of novel technologies in E. histolytica. This work will identify global transcriptional regulatory networks that control expression of amebic genes associated with virulence and provide a framework for characterization of the transcriptional machinery in Entamoeba histolytica. [unreadable] [unreadable] Entamoeba histolytica is an important pathogen and has an impact on human health on a global scale. The main disease manifestations are dysentery and liver abscesses. Although the majority of disease is in developing countries, this parasite can cause infections anywhere that water purification systems get adversely affected. Events such as the Tsunami or upheaval in the political and social infrastructure of a region can allow disease to emerge. We are interested in understanding the molecular mechanisms that the parasite uses to cause disease. [unreadable] [unreadable] [unreadable]